The treatment of a number of diseases can be achieved through gene therapy, where curative transgenes are introduced into a patient's cells by delivery with a vector of interest, for example viral, bacterial, mini-circle, etc. vectors. The delivered transgenes can integrate into the chromosomal DNA, replicate episomally or persist as non-replicating episomal elements in non-dividing cells. Depending on the properties of the transgene expression cassette, particular features of specific transgene integration sites and the state of the individual recipient cells, the transgenes are expressed with varying degree of efficiency. On some occasions, the transgenes are permanently silenced immediately after introduction, on other occasions transgene silencing occurs only after a certain period of adequate expression and on still other occasions transgene expression varies dramatically among the individual clones of transgene-harboring cells. Such variation is thought to be mainly due to the transgene's interaction with its immediate genetic neighborhood within the host genome. Stable long-term transgene expression in differentiating cells is particularly challenging. At a transcriptional level, the changing scenery of transcription initiation factor pools, chromatin re-modelling and DNA methylation events during differentiation contribute to the transiency of transgene expression.
Standard plasmid vectors composed of a transgene expression cassette and plasmid bacterial backbone (BB) DNA are able to express a high level of transgene product shortly after entering the cells, but the transgene product usually declines to very low or undetectable levels in a period of days even though the vector DNA is not lost. In fact, only very rare constructs in certain circumstances are able to express significant levels of transgene product for a prolonged period of time. There are a number of different factors (e.g., transgene product, mouse strain, and promoter) that may explain some of the variations in inter- and intralaboratory experimental results.
Early studies identified the nucleosome as the basic structural repeat unit of chromatin. It is composed of a nucleosome core containing 147 bp of DNA wrapped around a central histone octamer containing two molecules each of the four core histones (H2A, H2B, H3 and H4), and a “linker” DNA of characteristic length, which connects one nucleosome to the next. A single molecule of histone H1 (linker histone) is bound to the nucleosome at the point where the DNA enters and exits the core, and to the linker DNA. The DNA within the nucleosome core is protected from nucleases by the core histones, whereas the linker DNA is vulnerable to digestion. Thus, chromatin is composed of arrays of regularly spaced nucleosomes.
At a given moment in a cell population, there will be many possible chromatin states, including cells in which RNA polymerase II is initiating transcription at a nucleosome-free promoter, cells in which elongating RNA polymerase II is present at different places on the gene, causing local disruptions, and cells in which the gene is transiently in a non-transcribed state, or in the process of being remodeled. Thus, the combined effects of transcription and remodeling are expected to result in different chromatin structures at different times on the same gene. Many genes exhibit a sinusoidal nucleosome density profile, with peaks interpreted as positioned nucleosomes and troughs as linkers; many other genes exhibit more complex patterns that are difficult to interpret.
Methods of preventing transgene silencing from vectors introduced into cells are of great interest. The present invention addresses this issue.